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Supporting information
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Osmotic stress response in Acinetobacter baylyi: Identification of a glycine-betaine
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biosynthesis pathway and regulation of osmoadaptive choline uptake and glycine betaine
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synthesis through a choline-responsive BetI repressor.
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Anica Scholz, Julia Stahl, Veronique de Berardinis, Volker Müller and Beate Averhoff
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Appendix S1: Experimental procedures
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Betaine aldehyde dehydrogenase activity
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The betaine aldehyde dehydrogenase activity was determined by measuring the reduction of
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NADP+ to NADPH at 340 nm using a U-3000 spectrophotometer (Hitachi, Maidenhead, UK). Cells
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of A. baylyi and A. baylyi betB::kan were grown over night at 30 °C in 100 ml MM in the presence
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of 500 mM NaCl and 1 mM choline. Na-acetate was used as carbon and energy source. Cells
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were harvested at 7000 r.p.m. for 10 min and washed twice with potassium-phosphate buffer
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(0.1 M K2HPO4, pH 7.5). Cells were re-suspended in potassium-phosphate buffer (0.1 M K2HPO4,
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pH 7.5) and disrupted using a French pressure cell. Cell debris and unbroken cells were removed
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by centrifugation at 5000 r.p.m. for 20 min. The standard assay contained 100 mM K2HPO4 (pH
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7.5), 10 mM EDTA, 10 mM NADP+, 10 mM betaine aldehyde and various amounts of crude cell
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extract in a total reaction volume of 1 ml. The enzymatic reaction was started by the addition of
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betaine aldehyde, NADP+ or crude cell extract.
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Analysis of the transcriptional organization
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To analyze the transcriptional organization of the bet genes, cells were grown in 100 ml MM with
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500 mM NaCl and 1 mM choline. Na-acetate was used as carbon and energy source. Cells were
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harvested in the early exponential growth phase and total RNA was isolated using the „InviTrap®
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Spin Cell RNA Mini Kit“ (STRATEC Molecular GmbH, Berlin, Germany). cDNA was generated
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using M-MLVReverse Transcriptase (Promega, Mannheim, Germany). PCR using primers 1007-
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1008_For and 1007-1008_Rev, 1008-1009_For and 1008-1009_Rev, 1009-1010_For and 1009-
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1010_Rev, 1010-1011_For and 1010-1011_Rev, 1011-1012_For and 1011-1012_Rev, 1012-
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1013_For and 1012-1013_Rev, 1013-1014_For and 1013-1014_Rev (Table S1) was done using
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cDNA as template.
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Real-time PCR
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The qRT PCR analysis was performed with SYBR Green (Maxima SYBR Green qPCR Master
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Mix, Thermo Scientific, Waltham, MA, USA) as fluorescence dye. The reaction was monitored at
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521 nm and the data were analyzed using the 2-ΔΔCt – method (Livak and Schmittgen, 2001). As
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house-keeping gene the mdh gene (malate-dehydrogenase) was chosen (Sand et al., 2013).
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Primers that were generated for the qRT-PCR analysis are betA-for-RT and betA-rev-RT, betT1-
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for-RT and betT1-rev-RT, betT2-for-RT and betT2-rev-RT, mdh-RT-for and mdh-RT-rev (Table
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S1). Each primer pair amplifies a fragment 140 – 180 bp in length.
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Purification of BetI
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To overproduce and purify the potential regulatory protein BetI, the betI gene was cloned into the
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pET28a expression vector and transformed into E. coli BL21(DE3). The primers used to amplify
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the betI gene from genomic DNA of A. baylyi were BetI-His_for and BetI-His_rev (Table S1). The
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BetI-His_for primer was used to add a 6x His-tag and an NcoI restriction site to the 5’-end of the
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PCR product. The BetI-His_rev primer was used to add a NotI restriction site to the 3’-end. The
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PCR product was inserted into pET28a expression vector, resulting in pET28a-BetI-His. pET28a-
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BetI-His was transformed into E. coli BL21(DE3) and protein production was induced by addition
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of IPTG. Cells were harvested after 2.5 h and were resuspended in lysis buffer (10 mM imidazol,
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300 mM NaCl, 50 mM NaH2PO4, pH 7.5) prior to disruption by French pressure. The recombinant
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BetI-His protein was purified from the crude extract by affinity chromatography (Ni-NTA-agarose,
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Protino®, Macherey-Nagel, Düren, Germany). Therefore, the supernatant was incubated with 2 ml
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of Ni-NTA agarose at 4 °C for 1 h. The material was transferred into a column and the column was
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washed 3 times with washing buffer (50 mM and 120 mM imidazol, 300 mM NaCl, 50 mM
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NaH2PO4, pH 7.5) to remove non-specifically bound proteins. The recombinant BetI protein was
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eluted in 6 ml of elution buffer (800 mM imidazol, 300 mM NaCl, 50 mM NaH2PO4, pH 7.5). The
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elution fraction was concentrated to 0.5 ml using a Vivaspin 6 (MWCO: 10000, Sartorius Stedim
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biotech, Göttingen, Germany) and the protein was further purified by gelfiltration on Superdex 75
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(10/300 GL, GE Healthcare LifeScience, Munich, Germany) using ÄKTAprime plus (GE
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Healthcare LifeScience, Munich, Germany).
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Electrophoretic mobility shift assay
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To amplify the intergenic region of betI and betT1 primers emsa1_for and emsa1_rev were used.
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To amplify the intergenic region of betT1 and betT2 primers Promotor_XbaI and Promotor_BamHI
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(Table S1) were used. After amplification, part of the PCR product was labeled with α-[P32]-dCTP
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(specific activity 3000 Ci/mmol). Therefore 25 ng of PCR product were incubated at 99 °C for
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10 min and afterwards transferred onto ice. 2 µl of a hexa-nucleotide mixture, 3 µl of 1 mM dNTPs
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(dATP, dTTP, dGTP), 2 µl of 10 x Klenow-buffer (Thermo Scientific, Waltham, MA, USA) and 1 µl
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of Klenow-fragment (2 U/µl, Thermo Scientific, Waltham, MA, USA) were added and the total
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sample volume was adjusted to 15 µl with H2Odeion.. 5 µl of α-[P32]-dCTP (specific activity 3000
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Ci/mmol) were added and the sample was incubated at 37 °C for 30 – 45 min. The reaction was
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stopped by adding 2 µl of EDTA (200 mM, pH 8.0). For the electrophoretic mobility shift assay
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10 fmol of PCR product, 3.7 µl of glycerin (87 % [v/v]), 4 µl of 5 x EMSA buffer (250 mM MOPS,
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5 mM EDTA, 25 mM MgCl2, 5 mM DTT, 25% glycerin [v/v]) and different concentrations of purified
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BetI protein were mixed and the total sample volume was adjusted to 18 µl with H2Odeion..
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20000 cpm of α-[P32]-dCTP (specific activity 3000 Ci/mmol) labeled PCR product were added and
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the samples were incubated at 30 °C for 30 min. Choline or glycine betaine were added as
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indicated in the results. The samples were seperated by agarose gel electrophoresis and
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visualized by autoradiography of the dried gels.
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Statistical analysis
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Statistical analyses were performed with GraphPad Prism 4 (GraphPad Software Inc., San Diego,
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CA, USA). Values are presented as mean ± SEM.
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References (Appendix S1)
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Livak, K.j., and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time
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quantitative PCR and 2-Ct method. Methods 25: 402-408.
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Sand, M., Mingote, A.I., Santos, H., Müller, V., and Averhoff, B. (2013) Mannitol, a compatible
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solute synthesized by Acinetobacter baylyi in a two-step pathway including a salt-induced and
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salt-dependent mannitol-1-phosphate dehydrogenase. Environ Microbiol 15: 2187-2197.
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Table Legends
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Table S1
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Sequences of all primer used in this study
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Figure Legends
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Fig. S1
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Transcriptional organization of the bet gene cluster. The transcriptional organization of the bet gene cluster
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was analyzed by bridging analyses. P1-P7 mark the position of the primer pairs used (A). RNA was isolated
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and transcribed into cDNA, which served as template in qRT-PCRs. Results from qRT-PCR analyses using
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primer pairs P2 (B) and P3 (C) (Table S1) are shown. Lane 1: PCR product using cDNA as template, lane
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2: negative control with H2O, lane 3: positive control using genomic DNA as template. Shown is one
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representative result of two replicates.
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Fig. S2
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Purification of BetI and amplification of potential BetI-binding sites within the bet gene cluster. Coomassie
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stained 12% SDS-PAGE of purified BetI (A). Potential binding sites of BetI in the intergenic region of betI
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and betT1 and conserved -10 and -35 promotor region (B) and conserved -10 and -35 promotor site in the
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intergenic region of betT1 and betT2 (C). Bold arrows indicate the location of betI, betT1 and betT1 and
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their direction of transcription. Small arrows indicate primers used for the amplification of DNA fragments
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bridging the intergenic region of betI-betT1 and betT1-betT2, respectively. Small lower lines represent DNA
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fragments amplified with these primers.
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